Illudin analogs, uses thereof, and methods for synthesizing the same

ABSTRACT

This invention provides illudin derivatives, intermediates, preparation methods, pharmaceutical compositions and uses thereof. Specific examples include novel synthetic routes to prepare illudin derivatives and an illudin derivative having a positive optical rotation, which has therapeutic value.

TECHNICAL FIELD

This relates to illudin derivatives or analogs, intermediates,preparation methods, pharmaceutical compositions and uses thereof.

BACKGROUND

Illudins are a family of sesquiterpenes with antitumor antibioticproperties and are traditionally produced by various mushrooms. In theirisolated form, illudins show selective toxicity for myelocytic leukemiaand other malignant cells. Omphalotus species like O. olearius and O.illudens (Jack o' Lantern mushrooms), and O. nidiformis (Australianghost fungus) produce the natural forms of Illudins. Illudins are highlytoxic and have little therapeutic value in their natural forms.

Methods of manufacturing Illudin analogs have generally required theproduction of Illudin S from liquid growth of an O. illudins cellculture. FIG. 1 (prior art) shows the current semi-synthetic pathwayfrom Illudin S to hydroxymethylacylfulvene (HMAF) and (+)hydroxyureamethylacylfulvene. Although O. illudins cell lines have beendeveloped that produce a higher ratio of Illudin S to Illudin M, theproduction of this starting compound has been difficult in terms ofexpression yields, the time (e.g., >4 weeks of culture) required tobegin harvesting Illudin S, contamination with Illudin M, and othercomplications including difficulties with reproducibility. Thefermentation process can require the production, handling, andpurification of large quantities of Illudin S which is highly toxic andrepresents a biohazard in the manufacturing facility.

Accordingly, there has been a pressing need for improved Illudinsanalogs and methods to synthesize the same.

SUMMARY

This invention provides illudin derivatives, intermediates, preparationmethods, pharmaceutical compositions and uses thereof. Specific examplesinclude novel synthetic routes to prepare illudin derivatives and anilludin derivative having a positive optical rotation, which hastherapeutic value. The illudin derivatives react with DNA, which blocksthe transcription process, which can effectively treat cancers andinflammatory diseases.

Another embodiment provides a synthetic pathway to acylfulvene,irofulven (6-hydroxymethylacylfulvene), UMAF and other analogs ofcompound I or illudin. A method of synthesizing compounds of formula (I)in which R1, R2 and R3 are independently (C1-C4) alkyl, methyl, orhydroxyl. In addition, the specific embodiments include derivations ofenantiopure and racemic forms of acylfulvene, irofulven, and UMAF. Theracemic and positive (+) entantiomer of UMAF are novel compounds asdisclosed in the present invention.

DESCRIPTION OF THE FIGURES

FIG. 1 shows a prior art current semi-synthetic pathway from Illudin Sto hydroxymethyl acylfulvene (HMAF) and (+)hydroxyureamethylacylfulvene.

FIG. 2 shows one embodiment of this invention to prepare a pivitolintermediate or tertiary alcohol.

FIG. 3 shows another embodiment includes a method for synthesizing acompound into (±)-acylfulvene by two exemplary strategies.

FIG. 4 show that the (+) enantiomer is more toxic to the DU145 cellline.

FIG. 5 show that the PC3 cell line has similarly sensitive to bothenantiomeric forms of hydroxyureamethylacylfulvene,

FIG. 6 show that the (+) enantiomer is more toxic to the OVCAR3 cellline,

FIG. 7 show that the (+) enantiomer is more toxic to the SK-OV3 cellline.

FIG. 8 show that the HCC827 cell line has similarly sensitive to bothenantiomeric forms of hydroxyureamethylacylfulvene, and

FIG. 9 show that the (+) enantiomer is more toxic to the H1975 cellline.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in artto which the subject matter herein belongs. As used herein, thefollowing definitions are supplied in order to facilitate theunderstanding of the present invention.

As used herein, the terms a “patient,” “subject,” “individual,” and“host” refer to either a human or a non-human animal suffering from orsuspected of suffering from a disease or disorder associated withaberrant biological or cell growth activity.

The terms “treat” and “treating” such a disease or disorder refers toameliorating at least one symptom of the disease or disorder. Theseterms, when used in connection with a condition such as a cancer, referto one or more of impeding growth of the cancer, causing the cancer toshrink by weight or volume, extending the expected survival time of thepatient, inhibiting tumor growth, reducing tumor mass, reducing size ornumber of metastatic lesions, inhibiting the development of newmetastatic lesions, prolonging survival, prolonging progression-freesurvival, prolonging time to progression, and/or enhancing quality oflife.

The term “preventing” when used in relation to a condition or diseasesuch as cancer, refers to a reduction in the frequency of, or delay inthe onset of, symptoms of the condition or disease. Thus, prevention ofcancer includes, for example, reducing the number of detectablecancerous growths in a population of patients receiving a prophylactictreatment relative to an untreated control population, and/or delayingthe appearance of detectable cancerous growths in a treated populationversus an untreated control population, e.g., by a statistically and/orclinically significant amount.

The term “pharmaceutically acceptable” means that, which is useful inpreparing a pharmaceutical composition that is generally safe,non-toxic, and neither biologically nor otherwise undesirable andincludes that which is acceptable for veterinary as well as humanpharmaceutical use.

The term “stereoisomers” refers to any enantiomers, diastereomers, orgeometrical isomers of the compounds of formula (I) wherever they arechiral or when they bear one or more double bond. When the compounds ofthe formula (I) and related formulae are chiral, they can exist inracemic or in optically active form. Since the pharmaceutical activityof the racemates or stereoisomers of the compounds according to theinvention may differ, it may be desirable to use the enantiomers. Inthese cases, the end product or even the intermediates can be separatedinto enantiomeric compounds by chemical or physical measures known oremployed as such in the synthesis.

The term “therapeutic effect” refers to a beneficial local or systemiceffect in animals, particularly mammals, and more particularly humans,caused by administration of a compound or composition of the invention.The phrase “therapeutically-effective amount” means that amount of acompound or composition of the invention that is effective to treat adisease or condition caused by aberrant biological activity at areasonable benefit/risk ratio.

The therapeutically effective amount of such substance will varydepending upon the subject and disease condition being treated, theweight and age of the subject, the severity of the disease condition,the manner of administration and the like, which can readily bedetermined by one of skill in the art.

DETAILED DESCRIPTION

An embodiment of the present application provides illudin derivatives,intermediates, preparation methods, pharmaceutical compositions and usesthereof. Specific examples include novel synthetic routes to prepareilludin derivatives and an illudin derivative having a positive opticalrotation, which has therapeutic value. The illudin derivatives reactwith DNA, which blocks the transcription process, which can effectivelytreat cancers and inflammatory diseases.

One illustrative embodiment of this invention provides the compound withformula I.

The R₁, R₂ and R₃ are independently (C1-C4) alkyl, methyl, or hydroxyl

Another illustrative embodiment includes hydroxymethylacylfulvene (HMAF,Irofulven), which has the following formula II

Yet another illustrative embodiment includes(−)-HydroxyUreaMethylAcylfulvene, (UMAF), which has the followingformula III

One illustrative embodiment is characterized in that UMAF is theenantiomer, which exhibits at room temperature a positive opticalrotation in dichloromethane or methanol. UMAF has a positive opticalactivity or may be part of a racemic mixture/mixture of UMAF structures.A mixture can include the following structures:

This application provides methods of synthesizing compounds of formula(IV):

(−) 6-hydroxymethylacylfulvene, HMAF, Irofulven

Another embodiment provides a synthetic pathway to acylfulvene,irofulven (6-hydroxymethylacylfulvene), UMAF and other analogs ofcompound I or illudin. A method of synthesizing compounds of formula (I)in which R₁, R₂ and R₃ are independently (C1-C4) alkyl, methyl, orhydroxyl. In addition, the specific embodiments include derivations ofenantiopure and racemic forms of acylfulvene, irofulven, and UMAF. Theracemic and positive (+) entantiomer of UMAF are novel compounds asdisclosed in the present invention.

Referring now to FIG. 2 , one embodiment includes the steps of (1)converting 2-furfural using a Grignard reaction, to produce an alcoholof the below formula in which R₁ is a hydrogen atom, methyl group, alkylgroup, allyl or α-methylallyl group.

(2) Piancatelli rearrangement to the racemic cyclopentenone, and (3)protecting the alcohol group to furnish 9. In producing alcohols fromcarbonyl compounds by the Grignard reaction, a common practice is toprepare a Grignard reagent and then allow it to react with carbonylcompounds. The selection of appropriate protecting groups, can bereadily determined by one skilled in the art—exemplary protecting groupsinclude, but are not limited to, Silyl [trimethylsilane (TMS),tert-butyl, dimethylsilyl (TBS), acetates (Acetate (Ac), and benzoyl(Bz),] or benzyl [benzyl (Bn, para-methoxybenzyl (PMB)). Utilization ofa carbonyl-ylide dipolar-cycloaddition between cyclopentenone 9 anddiazoketone 10 provides the cycloadduct 11 which is transformed to 12via base-mediated elimination. Selective alkylation of the ketone overthe enone furnishes a pivitol intermediate: tertiary alcohol 13.removing the protecting group from the molecule (e.g., by base-mediatedelimination). Selective alkylation of the ketone over the enone providesthe tertiary alcohol.

Another embodiment provides a method for synthesizing acylfulvene (3),irofulven (4) and HydroxyUreaMethylAcylfulvene, (5) from a tertiaryalcohol. The tertiary alcohol may be a racemate or an enantiopure form,would commence with the alkylation of 2-furfural (6) with methylGrignard, followed by a Piancatelli rearrangement to the racemiccyclopentenone (±)-8 which would then be protected to furnish 9 (Scheme2).

Referring now to FIG. 3 , another embodiment includes a method forsynthesizing compound 13 into (±)-acylfulvene (3) by two exemplarystrategies (Scheme 3). In the one exemplary method, a Lewisacid-mediated elimination furnishes the dienone 14, which, afterreduction and elimination of the ketone moiety, produces the diol(±)-16. The oxidation of compound (±)-16 then can result in(±)-acylfulvene (3). In another exemplary method, reduction of compound13 yields alcohol 15, which after subjection to Lewis acidic conditionsproduces the diol (±)-16 and (±)-acylfulvene (3) is made in the samemanner. In another example, (±)-irofulven (4) and (±)-UMAF (5) can thenbe made via a similar sequence described in Scheme 1. The disclosedtransformation of 6 into acylfulvene (3), irofulven (4) and UMAF (5) maybe the shortest and most efficient synthesis of these compounds to date.

If enantiopure acylfulvene (3), irofulven (4) or UMAF (5) are desired, amixture or racemic intermediate (±)-16 can be purified via preparativechiral chromatography or other method known to those skilled in the artto produce both (+)-(16) and (−)-16 which can be used to synthesizeeither enantiomer of acylfulvene (3), Irofulven (4) or UMAF (5) (Scheme4). This represents a process for the enantioselective synthesis ofthese compounds.

As shown below, either enantiomer of acylfulvene (3), Irofulven (4) orUMAF (5) can also be synthesized via the known enzymatic resolution ofracemic (±)-9 to generate either (±)-9 or (−)-9, which can be convertedto either acylfulvene (3), Irofulven (4) or UMAF (5) via the proceduredescribed in Scheme 2 and 3 (Scheme 5).¹

Specific embodiments also feature pharmaceutical compositions containinga pharmaceutically acceptable carrier and any compound of Formulas (I),(II), (III) and other compounds shown above.

Specific embodiments also feature compositions and compounds, e.g.,UMAF, having reduced toxicity and side effects (including eye-relatedtoxicities). Other embodiments allow for methods for treatment takingadvantage of those properties.

Pharmaceutically acceptable salts of these compounds are alsocontemplated for the uses described herein. “Pharmaceutically acceptablesalt” refers to any salt of a compound of the invention which retainsits biological properties and which is not toxic or otherwiseundesirable for pharmaceutical use. Pharmaceutically acceptable saltsmay be derived from a variety of organic and inorganic counter-ions wellknown in the art and include. Such salts include: (1) acid additionsalts formed with organic or inorganic acids such as hydrochloric,hydrobromic, sulfuric, nitric, phosphoric, sulfamic, acetic,trifluoroacetic, trichloroacetic, propionic, hexanoic,cyclopentylpropionic, glycolic, glutaric, pyruvic, lactic, malonic,succinic, sorbic, ascorbic, malic, maleic, fumaric, tartaric, citric,benzoic, 3-(4-hydroxybenzoyl)benzoic, picric, cinnamic, mandelic,phthalic, lauric, methanesulfonic, ethanesulfonic,1,2-ethane-disulfonic, 2-hydroxyethanesulfonic, benzenesulfonic,4-chlorobenzenesulfonic, 2-naphthalenesulfonic, 4-toluenesulfonic,camphoric, camphorsulfonic,4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic, glucoheptonic,3-phenylpropionic, trimethylacetic, tert-butylacetic, lauryl sulfuric,gluconic, benzoic, glutamic, hydroxynaphthoic, salicylic, stearic,cyclohexylsulfamic, quinic, muconic acid and the like acids; or (2)salts formed when an acidic proton present in the parent compound either(a) is replaced by a metal ion, e.g., an alkali metal ion, an alkalineearth ion or an aluminum ion, or alkali metal or alkaline earth metalhydroxides, such as sodium, potassium, calcium, magnesium, aluminum,lithium, zinc, and barium hydroxide, ammonia or (b) coordinates with anorganic base, such as aliphatic, alicyclic, or aromatic organic amines,such as ammonia, methylamine, dimethylamine, diethylamine, picoline,ethanolamine, diethanolamine, triethanolamine, ethylenediamine, lysine,arginine, ornithine, choline, N,N′-dibenzylethylene-diamine,chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine,N-methylglucamine piperazine, tris(hydroxymethyl)-aminomethane,tetramethylammonium hydroxide, and the like. Pharmaceutically acceptablesalts further include, by way of example only, sodium, potassium,calcium, magnesium, ammonium, tetraalkylammonium and the like, and whenthe compound contains a basic functionality, salts of non-toxic organicor inorganic acids, such as hydrochloride, hydrobromide, tartrate,mesylate, besylate, acetate, maleate, oxalate and the like.

Pharmaceutical compositions of the invention comprise one or morecompounds of the invention and one or more physiologically orpharmaceutically acceptable carrier. The term “pharmaceuticallyacceptable carrier” refers to a pharmaceutically-acceptable material,composition or vehicle, such as a liquid or solid filler, diluent,excipient, solvent or encapsulating material, involved in carrying ortransporting any subject composition or component thereof. Each carriermust be “acceptable” in the sense of being compatible with the subjectcomposition and its components and not injurious to the patient. Someexamples of materials which may serve as pharmaceutically acceptablecarriers include: (1) sugars, such as lactose, glucose and sucrose; (2)starches, such as corn starch and potato starch; (3) cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7)talc; (8) excipients, such as cocoa butter and suppository waxes; (9)oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; (10) glycols, such as propyleneglycol; (11) polyols, such as glycerin, sorbitol, mannitol andpolyethylene glycol; (12) esters, such as ethyl oleate and ethyllaurate; (13) agar; (14) buffering agents, such as magnesium hydroxideand aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17)isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20)phosphate buffer solutions; and (21) other non-toxic compatiblesubstances employed in pharmaceutical formulations.

The compositions of the invention may be administered orally,parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. The term “parenteral”as used herein includes subcutaneous, intravenous, intramuscular,intra-articular, intra-synovial, intrasternal, intrathecal,intrahepatic, intralesional and intracranial injection or infusiontechniques. In some embodiments, the compositions of the invention areadministered orally, intraperitoneally or intravenously. Sterileinjectable forms of the compositions of this invention may be aqueous oroleaginous suspension. These suspensions may be formulated according totechniques known in the art using suitable dispersing or wetting agentsand suspending agents. The sterile injectable preparation may also be asterile injectable solution or suspension in a non-toxic parenterallyacceptable diluent or solvent. Among the acceptable vehicles andsolvents that may be employed are water, Ringer's solution and isotonicsodium chloride solution. In addition, sterile, fixed oils areconventionally employed as a solvent or suspending medium.

For this purpose, any bland fixed oil may be employed includingsynthetic mono- or di-glycerides. Fatty acids, such as oleic acid andits glyceride derivatives are useful in the preparation of injectables,as are natural pharmaceutically-acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions. These oilsolutions or suspensions may also contain a long-chain alcohol diluentor dispersant, such as carboxymethyl cellulose or similar dispersingagents that are commonly used in the formulation of pharmaceuticallyacceptable dosage forms including emulsions and suspensions. Othercommonly used surfactants, such as Tween, Spans and other emulsifyingagents or bioavailability enhancers which are commonly used in themanufacture of pharmaceutically acceptable solid, liquid, or otherdosage forms may also be used for the purposes of formulation.

The pharmaceutically acceptable compositions of this invention may beorally administered in any orally acceptable dosage form including, butnot limited to, capsules, tablets, aqueous suspensions or solutions. Inthe case of tablets for oral use, carriers commonly used include lactoseand corn starch. Lubricating agents, such as magnesium stearate, arealso typically added. For oral administration in a capsule form, usefuldiluents include lactose and dried cornstarch. When aqueous suspensionsare required for oral use, the active ingredient is combined withemulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added.

Alternatively, the pharmaceutically acceptable compositions of thisinvention may be administered in the form of suppositories for rectaladministration. These can be prepared by mixing the agent with asuitable non-irritating excipient that is solid at room temperature butliquid at rectal temperature and therefore will melt in the rectum torelease the drug. Such materials include cocoa butter, beeswax andpolyethylene glycols.

The pharmaceutically acceptable compositions of this invention may alsobe administered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well-known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other conventional solubilizingor dispersing agents.

The amount of the compounds of the present invention that may becombined with the carrier materials to produce a composition in a singledosage form will vary depending upon the host treated, the particularmode of administration. The compositions should be formulated so that adosage of between 0.01-100 mg/kg body weight/day of the inhibitor can beadministered to a patient receiving these compositions.

In terms of dosage, toxicity and therapeutic efficacy of compounds ofthe invention, including pharmaceutically acceptable salts anddeuterated variants, can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals. The LD₅₀ is thedose lethal to 50% of the population. The ED₅₀ is the dosetherapeutically effective in 50% of the population. The dose ratiobetween toxic and therapeutic effects (LD50/ED50) is the therapeuticindex. Compounds that exhibit large therapeutic indexes are preferred.While compounds that exhibit toxic side effects may be used, care shouldbe taken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects. Alternatively, asecond therapeutic agent can be administered to mitigate the toxic sideeffects of a primary treatment agent.

Data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds may lie within a range of circulating concentrations thatinclude the ED₅₀ with little or no toxicity. The dosage may vary withinthis range depending upon the dosage form employed and the route ofadministration utilized. For any compound, the therapeutically effectivedose can be estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound that achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

It should also be understood that a specific dosage and treatmentregimen for any particular patient will depend upon a variety offactors, including the activity of the specific compound employed, theage, body weight, general health, sex, diet, time of administration,rate of excretion, drug combination, and the judgment of the treatingphysician and the severity of the particular disease being treated. Theamount of a compound of the present invention in the composition willalso depend upon the particular compound in the composition.

Examples

In the following examples, the following abbreviations are used therein.

-   -   AcOH Acetic acid    -   DIBAL-H Diisobutylaluminium hydroxide    -   DIPEA diisopropylethylamine    -   DMSO Dimethylsulfoxide    -   IBX 2-iodoxybenzoic acid    -   L Liter    -   M Molar    -   MeOAc Methyl acetate    -   MeOH Methanol    -   Mmol millimole    -   MTBE Methy tert-butyl ether    -   NMR Nuclear Magnetic Resonance    -   TFA Trifluoroacetic acid    -   THE Tetrahydrofuran    -   TMS tetramethylsilane    -   TMSOTf trimethylsilyl trifluoromethanesulfonate

Unless otherwise noted, solvents and reagents were used withoutpurification. CH₂Cl₂ and MeCN were stored over 4 Å molecular sieves.Volatile solvents were removed under reduced pressure using a Buchirotary evaporator. Thin layer chromatography (TLC) was performed onglass-backed precoated silica gel plates (0.25 mm thick with 60 F254)and were visualized using one or more of the following manners: UV light(254 nm), staining with I₂ impregnated silica, KMnO₄ or Ceric AmmoniumMolybdate (CAM). Flash chromatography was performed using the BiotageIsolera One using pre-loaded Silicycle 25 g high performance (14-40 μM)columns. ¹H nuclear magnetic resonance (NMR) spectra were obtained at400 MHz as indicated as solutions in CDCl3 with 0.05% v/vtetramethylsilane (TMS) unless indicated otherwise. ¹³C-NMR wereobtained at 100 MHz as shown in the indicated deuterated solvent.Chemical shifts are reported in parts per million (ppm, δ), andreferenced to TMS, and coupling constants are reported in Hertz (Hz).Spectral splitting patterns are designated as s, singlet; d, doublet; t,triplet; q, quartet; quint, quintuplet; sex, sextet; sept, septuplet; m,multiplet; comp, overlapping multiplets of magnetically nonequivalentprotons; br, broad; and app, apparent.

1-acetylcyclopropanecarboxylic acid. tert-butyl 3-oxobutanoate (339 g,350 mL, 2.146 mol) was added to a vigorously stirred mixture of K₂CO₃(1186 g, 8.58 mol) and DMSO (3.5 L) in a flask. The mixture was stirredfor 10 minutes whereupon neat 1,2-dibromoethane (806 g, 370 mL, 4.29mol) was added and the mixture was stirred overnight. The reactionmixture was then diluted with H₂O (2 L) and the mixture was extractedwith MTBE (3×500 mL). The combined organic fractions were washed with10% brine solution (4×200 mL) and the organic layer was dried overNa₂SO₄ and concentrated to an oil under reduced pressure. Neat TFA (486g, 328 mL, 4.29 mol) was added to the oil and the mixture was stirred atroom temperature overnight. The TFA was removed under reduced pressureand an aqueous solution of 20% (w/w) NaOH was added until the pH of theaqueous layer was <11. The mixture was then washed with MTBE (3×200 mL)and the aqueous layer was pH adjusted to >2 with an aqueous solution of20% (w/w) H2SO4 and the mixture was extracted with CH₂Cl₂ (3×200 mL) andthe combined organic layers were dried over Na₂SO₄ and concentratedunder reduced pressure to recover 182 g (66%) of crude1-acetylcyclopropanecarboxylic acid as an orange oil.

1-(furan-2-yl)ethan-1-ol. (7) A three neck round bottom flask (22 L) waswashed with anhydrous THF (100 mL), evacuated and filled with nitrogen(3×). The flask was then charged with THF (1500 mL) and furfural (600.0g, 547.2 mL, 6.244 mol) and the solution was cooled to 0° C. with an icebath and kept under a nitrogen atmosphere. A solution of methylmagnesiumchloride (2289 mL, 3 M, 6.838 mol) in THF was added slowly dropwise over˜180 min using a cannula using nitrogen pressure, carefully maintaininga reaction temperature less than 10° C. The mixture was allowed to coolto 0° C. and stirred for an additional 30 min before carefully quenchingwith 1N aq. HCl (1000 mL) with vigorous stirring, during which copiousamounts of solids formed and the reaction mixture solidified. Water(2000 mL) was then added and the solids were agitated mechanically tobreak up the solids before adding an additional 1N aq. HCl (5000 mL)while carefully maintaining an internal temperature ≤10° C. The mixturewas extracted with MTBE (3×1500 mL) and the organics were combined,washed with water (1000 mL), brine (1500 mL) then dried over sodiumsulfate. The organic layer was then concentrated under reduced pressureby rotary evaporator to recover 678 g (96%) of crude1-(furan-2-yl)ethan-1-ol as a ruby red liquid, which was taken ondirectly to the next step.

(±)-4-hydroxy-5-methylcyclopent-2-en-1-one. (8) A 2000 mL round bottomflask equipped with a reflux condenser and magnetic stir bar was chargedwith deionized water (1600 mL) and 1-(2-furyl)ethanol (7) (130 g, 1159mmol). The reaction mixture was purged with nitrogen and the mixture wasbrought to reflux and was heated for 120 min with vigorous stirring. Thereaction mixture was then allowed to cool to ambient temperature and theaqueous solution was decanted from the brown oily resin. The aqueouslayer was then washed with a mixture of MTBE and hexanes (1:1, 3×250 mL)and the organic extracts were discarded. Sodium chloride (250 g) wasthen added to the aqueous solution and extracted with EtOAc (3×250 mL).The combined organic extracts were dried over sodium sulfate, filtered,and concentrated under reduced pressure on a rotary evaporator torecover 8.0 g (6%) of (±)-4-hydroxy-5-methylcyclopent-2-en-1-one (8) asa pale yellow oil.

(±)-4-(Methoxymethoxy)-5-methylcyclopent-2-en-1-one. (9) A flask wascharged with (±)-4-hydroxy-5-methylcyclopent-2-en-1-one (8) (1.5 g,13.38 mmol) CH₂Cl₂ (80 mL), and diisopropylethylamine (DIPEA) (5.19,6.99 mL g, 40.14 mmol) and the mixture was purged with nitrogen andcooled to 0° C. with an ice bath. Chloromethyl methyl ether (3.14 M inMeOAc; 8.51 mL) was added dropwise via syringe over 20 min. The reactionwas stirred for an additional hour at 0° C. before being allowed toslowly warm up to ambient temperature and stirring overnight. Thereaction mixture was then diluted with CH₂Cl₂ (50 mL), quenched with 10%(aq) NH₄Cl (50 mL) and the organics were separated. The aqueous layerwas extracted with CH₂Cl₂ (50 mL) and the combined organic extracts weredried over sodium sulfate and concentrated under reduced pressure byrotary evaporator. The crude residue was purified by flashchromatography on silica gel (column: 40 g silicycle; Eluent: gradientCH₂Cl₂ 0→100% in hexane) to recover 10.5 g (84%) of(±)-4-(Methoxymethoxy)-5-methylcyclopent-2-en-1-one (9) as a pale yellowoil.

Diazoketone. (10) Oxalyl chloride (39.6 g, 26.4 mL, 312 mmol) was addedto a solution of 1-acetylcyclopropanecarboxylic acid (20 g, 156 mmol)and DMF (110 mg, 0.12 mL, 1.5 mmol) in CH₂Cl₂ (200 mL) at roomtemperature and stirred for two hours. The reaction mixture wasconcentrated to an oil under reduced pressure and dissolved in anhydrousCH₂Cl₂ (300 mL) and cooled to −78° C. (dry ice/acetone bath). Neat2,6-lutidine (19.3 g, 21.0 mL, 180 mmol) was added, followed by asolution of TMSCHN₂ (2.0 M, 200 mL, 300 mmol) in hexanes. The coolingbath was removed and the mixture was allowed to warm to room temperatureand stir overnight. The mixture was concentrated to an oil under reducedpressure before adding MTBE (200 mL) and storing in the fridge for 30min. The mixture was filtered through Celite (50 g), concentrated to anoil and purified on a silica plug (350 g, elute with 1000 mL CH₂Cl₂) andconcentrating the eluent to recover 29.3 g of a deep red-brown oil whichcontained 11.23 g diazoketone (10) (49%) by NMR using mesitylene as aninternal standard. The material was taken directly on to the next step.

Cycloadduct. (11) A stirring solution of diazoketone (10) (11.23 g, 73.8mmol) and (±)-4-(Methoxymethoxy)-5-methylcyclopent-2-en-1-one (9) (5.76g, 36.9 mmol) in CH₂Cl₂ was evacuated and filled with nitrogen (3×)before adding solid Rh₂(OAc)₄ (42 mg, 0.095 mmol) at room temperatureand stirring overnight. The reaction was then concentrated under reducedpressure and purified by flash chromatography on silica gel (column: 120g silicycle; Eluent: gradient EtOAc 0→80% in hexane) to recover 10.8 g(96%) by NMR using mesitylene as an internal standard. of Cycloadduct(11) as a waxy orange solid.

¹H-NMR (400 MHz) δ 4.96 (s, 1H), 4.80 (d, J=7.2 Hz, 1H), 4.74 (d, J=7.2Hz, 1H), 3.96 (dd, J=8.0, 11.2 Hz, 1H), 3.45 (s, 3H), 2.93 (t, J=7.6 Hz,1H), 2.67 (d, J=6.8 Hz, 1H), 2.59 (dq, J=6.4, 11.6 Hz, 1H), 1.30 (ddd,J=4.0, 6.8, 9.6 Hz, 1H), 1.20 (s, 3H). 1.16 (ddd, J=4.0, 6.4, 8.4 Hz,1H). 1.12 (s, 3H), 1.07 (ddd, J=4.4, 7.6, 9.6 Hz, 1H), 0.73 (ddd, J=4.0,7.2, 9.6 Hz, 1H); ¹³C-NMR (100 MHz) δ 212.8, 211.8, 96.2, 87.4, 81.5,79.9, 59.5, 50.2, 44.1, 39.0, 14.2, 13.8, 12.4, 11.2.

NMR assignments: ¹H-NMR (400 MHz) δ 4.96 (C9-H), 4.80 (C7-H), 4.74(C7-H), 3.96 (C4-H), 3.45 (C8-H), 2.93 (C3-H), 2.67 (C2-H), 2.59 (C5-H),1.30 (C14 or C15-H), 1.20 (C13-H). 1.16 (C14 or C15-H). 1.12 (C6-H),1.07 (C14 or C15-H), 0.73 (C14 or C15-H); ¹³C-NMR (100 MHz) δ 212.8(C1), 211.8 (C10), 96.2 (C7), 87.4 (C12), 81.5 (C9), 79.9 (C4), 59.5(C2), 50.2 (C8, C5), 44.1 (C3), 39.0 (C11), 14.2 (C14 or C15), 13.8(C13), 12.4 (C14 or C15), 11.2 (C6).

Methyladduct. Starting material (11) (5.6 g, 19.97 mmol) was dissolvedin dry THF (80 mL) and cooled to −78° C. with a dry ice/acetone bath.The solution was put under vacuum and back-filled with nitrogen (2times), whereupon a solution of MeMgCl (10 mL, 3 M in THF, 30 mmol; 1.5eq) was added dropwise and the reaction was stirred at −78° C. for 3.5 hbefore quenching with acetic acid (1.3 mL). The reaction was warmed toroom temperature, diluted with DCM (350 mL) and water (250 mL). Theresulting mixture was separated. Aqueous phase was extracted with DCM(100 mL) and the combined organic extracts were washed with 20% aq. NaCl(100 mL) and dried over Na₂SO₄ and concentrated to an oil. The oil waspurified via silica gel column chromatography (column: 40 g silicycle;Eluent: gradient EtOAc 0→45% in hexane) to recover 778 mg (13.2%) ofmethyladduct target product as a white crystals, along with 2.23 g(39.8%) of starting material, 762 mg (12.9%) of second isomer and 833 g(13.5%) of bis methyl adduct for a total mass balance of 92.6%.

Cyclopentenone. (13) Potassium carbonate (704 mg, 5.09 mmol; 1.0 eq) wasadded to a solution of starting material (1.50 g, 5.09 mmol) in MeOH (60mL) and the mixture was stirred at room temperature for 2 h beforequenching with 10% aq. NH₄Cl (10 mL) and diluted with EtOAc 300 mL. Theresulting mixture was washed with 20% NaCl 100 mL. Organic layer wasseparated and washed with 20% NaCl (50 mL). The organic extract wasdried over Na₂SO₄, concentrated to an oil and purified via silica gelcolumn chromatography (column: 25 g silicycle; Eluent: gradient EtOAc0→65% in hexane) to recover 974 mg (82%) of alkene Cyclopentenone (13)as a pale yellow crystals.

Dieneone. (14) Neat TMSOTf (7.72 mL, 9.48 g, 42.68 mmol) was added to asolution of bicycle (13) (2.0 g, 8.54 mmol) and 2,6-lutidine (7.46 mL,6.86 g, 64.05 mmol) in dry CH₂Cl₂ (100 mL) at 0° C. and the solution wasstirred for 4 hours under nitrogen atmosphere, whereupon the reactionwas quenched with MeOH (4 mL) and the reaction mixture was concentratedto a red-orange oil under reduced pressure. The crude oil was suspendedin MeOH (50 mL) and NH₄F (6.32 g, 170.8 mmol) and AcOH (8.55 mL, 8.97 g,149.45 mmol) was added and the suspension was stirred at roomtemperature overnight. The reaction was then diluted with H₂O (250 mL),extracted with EtOAc (2×150 mL) and the organic layer was washed with 5%(aq) citric acid monosodium salt (2×100 mL), dried over Na₂SO₄,concentrated to an oil and purified via silica gel column chromatography(column: 25 g silicycle; Eluent: gradient EtOAc 0→55% in hexane) torecover 774 mg (39%) of the dieneone product (14) as a yellowish solid.

Diol. (16) A solution of dienone (14) (770 mg, 3.28 mmol) in dry CH₂Cl₂(50 mL) was cooled to −78° C. with a dry ice/acetone bath before addinga solution of DIBAL-H (13.69 mL, 1.2 M in toluene, 16.43 mmol) inhexanes. The solution was stirred for 0.5 hours, whereupon the reactionwas quenched with MeOH (4 mL) and the reaction mixture was concentratedto an oil under reduced pressure. In a separate flask, H₂O (1 mL)followed by H₃PO₄ (85% w/w in H₂O, 0.35 mL) was added to a vigorouslystirred suspension of silica (10 g) in CH₂Cl₂ (50 mL) and the mixturewas stirred at room temperature for 2 h. The solvent was then removedunder reduced pressure until the silica was free-flowing. The crude oilfrom the DIBAL-H reduction was dissolved in EtOAc (200 mL) before addingH₃PO₄-doped hydrated silica (6.2 g) and the suspension was stirredvigorously for 3 h. The reaction was then quenched with TEA (430 μL, 312mg, 3.08 mmol) and filtered. The resulting solution was concentrated toan oil and purified via silica gel column chromatography (column: 4 gsilicycle; Eluent: gradient EtOAc 0→50% in hexane) to recover 83 mg(12%) of compound diol (16) as a yellow solid.

(−)-Acylfulvene. (3) Diol (16) (38 mg, 0.174 mmol) was driedazeotropically by dissolving in toluene (5 mL) and concentrating underreduced pressure before adding dry DMSO (6 mL) followed by IBX 45 wt. %(216 mg, 0.348 mmol) and the suspension was stirred at room temperaturefor 2 h. The mixture was then diluted with H₂O (15 mL), extracted withEtOAc (2×10 mL), dried over Na₂SO₄, concentrated to an oil and purifiedvia silica gel column chromatography (column: 12 g silicycle; Eluent:gradient EtOAc 0→50% in hexane) to recover 37 mg (98%) of(−)-Acylfulvene (3) as a yellow gum.

¹H-NMR (400 MHz) δ 7.16 (d, J=0.8 Hz, 1H), 6.43 (quint, J=1.6 Hz, 1H),3.93 (bs, 1H), 2.15 (d, J=1.2 Hz, 3H), 2.00 (s, 3H), 1.52 (ddd, J=4.0,6.4, 9.6 Hz, 1H), 1.38 (s, 3H), 1.29 (ddd, J=4.8, 6.4, 9.6 Hz, 1H), 1.07(ddd, J=5.2, 7.2, 9.6 Hz, 1H), 0.71 (ddd, J=4.0, 7.2, 9.6 Hz, 1H).

NMR assignments: ¹H-NMR (400 MHz) δ 7.16 (C4-H), 6.43 (C1-H), 3.93(O—H), 2.15 (C6-H), 2.00 (C11-H), 1.52 (C12 or C13-H), 1.38 (C14-H),1.29 (C12 or C13-H), 1.07 (C12 or C13-H), 0.71 (C12 or C13-H).

(+)-Acylfulvene. (3) Diol (30 mg, 0.137 mmol) was dried azeotropicallyby dissolving in toluene (5 mL) and concentrating under reduced pressurebefore adding dry DMSO (5 mL) followed by IBX 45 wt. % (171 mg, 0.275mmol) and the suspension was stirred at room temperature for 2 h. Themixture was then diluted with H₂O (15 mL), extracted with EtOAc (2×10mL), dried over Na₂SO₄, concentrated to an oil and purified via silicagel column chromatography (column: 12 g silicycle; Eluent: gradientEtOAc 0→50% in hexane) to recover 30 mg (100%) of (+)-Acylfulvene as ayellow gum.

¹H-NMR (400 MHz) δ 7.16 (d, J=0.8 Hz, 1H), 6.43 (quint, J=1.6 Hz, 1H),3.93 (bs, 1H), 2.15 (d, J=1.2 Hz, 3H), 2.00 (s, 3H), 1.52 (ddd, J=4.0,6.4, 9.6 Hz, 1H), 1.38 (s, 3H), 1.29 (ddd, J=4.8, 6.4, 9.6 Hz, 1H), 1.07(ddd, J=5.2, 7.2, 9.6 Hz, 1H), 0.71 (ddd, J=4.0, 7.2, 9.6 Hz, 1H).

(−)-Irofulven. (4) A suspension of paraformaldehyde (231 mg, 7.7 mmol asmonomer) in 2M (aq) H₂SO₄ (4 mL) was heated to 90° C. for 30 min andcooled to room temperature before adding acetone (4 mL) and a solutionof (−)-Acylfulvene (3) (37 mg, 0.171 mmol) in acetone (1 mL) and themixture was stirred for 48 h at room temperature. The reaction was thendiluted with H₂O (25 mL), extracted with CH₂Cl₂ (3×15 mL), and thecombined organic extracts were washed with NaHCO₃ (aq) (15 mL) then H₂O(15 mL), water wash pH ˜7. The organic extracts were then dried overNa₂SO₄, concentrated to an oil and purified via silica gel columnchromatography (column: 12 g silicycle; Eluent: gradient EtOAc 0→65% inhexane) to recover 24.6 mg (58%) of (−)-Irofulven (4) as a yellow gumand 8.6 mg (23%) of recovered (−)-Acylfulvene.

¹H-NMR (400 MHz) δ 7.09 (s, 1H), 4.67 (d, J=12.4 Hz, 1H), 4.63 (d,J=12.4 Hz, 1H), 3.90 (bs, 1H), 2.19 (s, 3H), 2.15 (s, 3H), 1.49 (ddd,J=4.0, 6.0, 9.6 Hz, 1H), 1.38 (s, 3H), 1.36 (ddd, J=5.2, 6.4, 9.6 Hz,1H), 1.08 (ddd, J=4.8, 7.2, 9.6 Hz, 1H), 0.72 (ddd, J=4.0, 7.6, 10.0 Hz,1H).

NMR assignments: ¹H-NMR (400 MHz) δ 7.09 (C4-H), 4.67 (C15-H), 4.63(C15-H), 3.90 (O—H), 2.19 (C6-H), 2.15 (C11-H), 1.49 (C12 or C13-H),1.38 (C14-H), 1.36 (C12 or C13-H), 1.08 (C12 or C13-H), 0.72 (C12 orC13-H).

(+)-Irofulven. (4) A suspension of paraformaldehyde (186 mg, 6.21 mmolas monomer) in 1M (aq) H₂SO₄ (9 mL) was heated to 90 HC for 30 min andcooled to room temperature before adding acetone (9 mL) and a solutionon (+)-Acylfulvene (3) (30 mg, 0.138 mmol) in acetone (3 mL) and themixture was stirred for 72 h at room temperature. The reaction was thendiluted with H₂O (30 mL), extracted with CH₂Cl₂ (3×15 mL) and thecombined organic extracts were washed with NaHCO₃ (aq) (15 mL) then H₂O(15 mL), water wash pH ˜7. The organic extracts were then dried overNa₂SO₄, concentrated to an oil and purified via silica gel columnchromatography (column: 12 g silicycle; Eluent: gradient EtOAc 0-65% inhexane) to recover 12.7 mg (37%) of (+)-Irofulven as a yellow gum and12.4 mg (41%) of recovered (+)-Acylfulvene (3).

¹H-NMR (400 MHz) δ 7.09 (s, 1H), 4.67 (d, J=12.4 Hz, 1H), 4.63 (d,J=12.4 Hz, 1H), 3.90 (bs, 1H), 2.19 (s, 3H), 2.15 (s, 3H), 1.49 (ddd,J=4.0, 6.0, 9.6 Hz, 1H), 1.38 (s, 3H), 1.36 (ddd, J=5.2, 6.4, 9.6 Hz,1H), 1.08 (ddd, J=4.8, 7.2, 9.6 Hz, 1H), 0.72 (ddd, J=4.0, 7.6, 10.0 Hz,1H).

NMR assignments: ¹H-NMR (400 MHz) δ 7.09 (C4-H), 4.67 (C15-H), 4.63(C15-H), 3.90 (O—H), 2.19 (C6-H), 2.15 (C11-H), 1.49 (C12 or C13-H),1.38 (C14-H), 1.36 (C12 or C13-H), 1.08 (C12 or C13-H), 0.72 (C12 orC13-H).

(−)-hydroxyureamethylacylfulvene. (5) Hydroxyurea (37 mg, 0.487 mmol)was added to a solution of (−)-Irofulven (4) (24 mg, 0.097 mmol) in amixture of acetone (1.5 mL) and 2M (aq) H₂SO₄ (1.5 mL) and the mixturewas stirred for 24 h at room temperature before diluting with H₂O (15mL) and EtOAc (15 mL), organics were separated and aqueous layer wasextracted with EtOAc (2×10 mL). The combined organic extracts werewashed with 5% NaHCO₃ (aq) (10 mL) the brine (10 mL). The organicextracts were then dried over Na₂SO₄, concentrated to an oil andpurified via silica gel column chromatography (column: 12 g silicycle;Eluent: gradient EtOAc 10→95% in hexane) to recover 18.9 mg (61%) of(−)-LP-184 (5) as a yellow-orange solid.

¹H-NMR (400 MHz) δ 7.03 (s, 1H), 6.86 (s, 1H), 5.37 (bs, 2H), 4.80 (d,J=14.8 Hz, 1H), 4.52 (d, J=14.5 Hz, 1H), 3.84 (bs, 1H), 2.17 (s, 3H),2.08 (s, 3H), 1.47 (ddd, J=4.0, 6.4, 10.0 Hz, 1H), 1.35 (ddd, J=5.2,6.4, 9.6 Hz, 1H), 1.35 (s, 3H), 1.34 (ddd, J=5.2, 7.6, 9.6 Hz, 1H), 0.68(ddd, J=4.0, 7.6, 10.0 Hz, 1H).

(+)-hydroxyureamethylacylfulvene. (5) Hydroxyurea (7.5 mg, 0.098 mmol)was added to a solution of (+)-Irofulven (4) (12 mg, 0.049 mmol) in amixture of acetone (1 mL) and 2M (aq) H₂SO₄ (1 mL) and the mixture wasstirred for 24 h at room temperature before diluting with H₂O (15 mL)and EtOAc (15 mL), organics were separated and aqueous layer wasextracted with EtOAc (2×10 mL). The combined organic extracts werewashed with NaHCO₃ (aq) (10 mL) the brine (10 mL). The organic extractswere then dried over Na₂SO₄, concentrated to an oil and purified viasilica gel column chromatography (column: 12 g silicycle; Eluent:gradient EtOAc 10→95% in hexane) to recover 6.6 mg (44%) of (+)-LP-184(5) as a yellow solid and 3.0 mg (25%) of recovered (+)-Irofulven (4).

¹H-NMR (400 MHz) δ 7.03 (s, 1H), 6.86 (s, 1H), 5.37 (bs, 2H), 4.80 (d,J=14.8 Hz, 1H), 4.52 (d, J=14.5 Hz, 1H), 3.84 (bs, 1H), 2.17 (s, 3H),2.08 (s, 3H), 1.47 (ddd, J=4.0, 6.4, 10.0 Hz, 1H), 1.35 (ddd, J=5.2,6.4, 9.6 Hz, 1H), 1.35 (s, 3H), 1.34 (ddd, J=5.2, 7.6, 9.6 Hz, 1H), 0.68(ddd, J=4.0, 7.6, 10.0 Hz, 1H).

Cytotoxicity. The growth-inhibition activities of both purified UMAF(also referred to as UMAF in this example) optical enantiomers werestudied in a standard 96-well cell-based assay using representative celllines from prostate, ovarian, and lung cancers. The concentrations thatcause a 50% inhibition of cell growth (GI₅₀) were determined using aluminescent vital assay reagent (CellTiter-Glo®, Promega Corporation).The growth inhibition curves as a function of UMAF concentration areshown from a representative assay. FIGS. 4, 6, 7, and 9 show that the(+) enantiomer is more toxic to DU145, OVCAR3, SK-OV3, and H1975 celllines, respectively. FIGS. 5 and 8 show that the PC3 and HCC827 celllines are similarly sensitive to both enantiomeric forms of thecompound. The GI₅₀ concentrations of the two forms for the six celllines are shown below.

Activity of UMAF on cancer cell lines* Prostate Ovarian Lung DU145 PC3OVCAR-3 SK-OV3 HCC827 H1975 (+)-UMAF 34.8 1,120 254 554 897 287 (−)-UMAF506 824 1,820 2,400 1,640 6,320 *Nanomolar concentrations of UMAF thatinhibits 50% cell growth in a 3-day assay

What is claimed:
 1. A process for synthesizing compounds having theformula (III):

wherein Ri, R₂ and R₃ are independently (C1-C4) alkyl, methyl, orhydroxyl, comprising the steps of selecting a cyclopentenone having analcohol group; protecting the alcohol group; reacting the protectedcyclopentenone with a diazoketone to form a cycloadduct; and reducingthe cycloadduct.
 2. The method according to claim 1, wherein thecyclopentenone of step a is selected from the group consisting ofsubstituted cyclopentenones.
 3. The method according to claim 1, whereinthe cleaving of the oxy group is achieved through the use of a suitableacid or base.
 4. The process according to claim 1, wherein thediazoketone is derived from a diazo compound and a ketone.
 5. Theprocess of claim 1, wherein R1, R2, and R3 are each independentlyselected from (C1-C4) alkyl groups.
 6. The process of claim 1, furthercomprising oxidizing the cleaved cycloadduct to obtainhydroxyureamethylacylfulvene (+).
 7. The method according to claim 6,further comprising isolating hydroxyureamethylacylfulvene (+) after stepg) using standard purification techniques.
 8. A method for synthesizingenantiopure acylfulvene, hydroxymethyl acylfulvene (HMAF), andhydroxyureamethylacylfulvene (UMAF) utilizing an enzymatic resolution ofCompound 9 (Scheme 5).


9. A method of synthesizing hydroxyureamethylacylfulvene (+) comprisingthe steps of: a) selecting a cyclopentenone having an alcohol group; b)protecting the alcohol group of the selected cyclopentenone; c) reactingthe protected cyclopentenone with a diazoketone to form a cycloadduct;d) Reducing the cycloadduct obtained in step c; e) Alkylating a ketonegroup of the reduced cycloadduct; f) Cleaving an oxy group of thealkylated cycloadduct; and g) Oxidizing the cleaved cycloadduct toobtain hydroxyureamethylacylfulvene (+).
 10. The method according toclaim 9, wherein the cyclopentenone of step a is selected from the groupconsisting of substituted and unsubstituted cyclopentenones.
 11. Themethod according to claim 9, wherein the protecting group used in step bis selected from the group consisting of acetyl, benzyl,tert-butyldimethylsilyl (TBDMS), and tert-butoxy carbonyl groups. 12.The method according to claim 9, wherein the cleaving of the oxy groupin step f is achieved through the use of a suitable reagent, such as anacid or base.
 13. The method according to claim 9, wherein thealkylating agent used in step 5 is selected from the group consisting ofalkyl halides, alkyl sulfonates, and alkyl tosylates.
 14. The methodaccording to claim 9, wherein the alkylating agent of step e) is analkyl halide.
 15. The method according to claim 9, further comprisingisolating hydroxyureamethylacylfulvene (+) after step g by standardpurification techniques.